Use of protein kinase C inhibitors to enhance the clinical efficacy of oncolytic agents and radiation therapy

ABSTRACT

A method for treating neoplasms is disclosed, particularly using the beta-isozyme selective PKC inhibitor, (S)-3,4-[N, N&#39;-1,1&#39;-((2&#39;&#39;-ethoxy)-3&#39;&#39;&#39;(O)-4&#39;&#39;&#39;-(N,N-dimethylamino)-butane)-bis-(3,3&#39;-indolyl)]-1(H)-pyrrole-2,5-dione or one of its salts, such PKC inhibitors enhance the clinical efficacy of oncolytic agents and radiation therapy.

This application is a continuation of U.S. Ser. No. 08/841,738 filedApr. 30, 1997 which now U.S. Pat. No. 6,232,299 claims the prioritybenefits of the U.S. Provisional application Ser. No. 60/016,658 filedMay 1, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is broadly directed to a method for enhancinganti-neoplasm effects of chemotherapies and radiation therapies with PKCinhibitors. The present invention is particularly directed to the use ofProtein Kinase C (PKC) inhibitors, especially a particular class ofisozyme selective PKC inhibitors in combination with an oncolytic agentor γ-irradiation to enhance their anti-neoplasm effects in treatment ofneoplasms.

2. Description of Related Art

Therapeutic treatments have been developed over the years to treatneoplasms. There are two major approaches to treat neoplasms: 1)chemotherapy employing oncolytic agents, and 2) radiation therapy, e.g.,γ-irradiation. Oncolytic agents and γ-irradiation cause cytotoxiceffects, preferentially to tumor cells, and cause cell death.

Studies have shown that γ-irradiation and certain groups of oncolyticagents assert their cytotoxic effects by activating programmed celldeath or apoptosis. A balance between the activities of apoptotic andantiapoptotic intracellular signal transduction pathways is importanttowards a cell's decision of undergoing apoptosis in response to theabove mentioned chemotherapy as well as radiation therapy.

PKC inhibitors has been proposed for cancer therapy, e.g. see U.S. Pat.No. 5,552,391, and PKC activities have been indicated to exertantiapoptotic effects, especially in response to radiation therapies,e.g., γ-irradiation. In particular, studies have shown that activationof PKC inhibits apoptosis induced by anti-neoplasm agents such as Ara-c,2-chloro-2-deoxyadenosine, 9-β-D-arabinosyl-2-fluoroadenine, andγ-irradiation therapy. There also have been indications that downregulation of PKC activities in tumor cells enhances apoptosisstimulated by oncolytic agents. PKC activation has been shown toattenuate γ-irradiation induced cell death.

There is a need in the art to develop therapeutic agents which enhancethe apoptotic signal transduction pathways in cells and thereby enhancethe clinical efficacy of oncolytic agents and radiation therapy.

SUMMARY OF INVENTION

It is an object of the invention to provide methods for treating aneoplasm.

It is another object of the invention to provide methods for enhancingan anti-neoplasm effect of an oncolytic agent.

It is still another object of the invention to provide methods forenhancing anti-neoplasm effects of radiation therapy.

These and other objects of the invention are provided by one or more ofthe embodiments described below.

In one embodiment of the invention there is provided a method fortreating a neoplasm which comprises administrating to a mammal in needof such treatment an oncolytic agent or γ-irradiation in combinationwith a protein kinase C inhibitor.

In still another embodiment of the invention there is provided a methodfor enhancing an anti-neoplasm effect of chemotherapy and radiationtherapy which comprises administrating a protein kinase C inhibitor incombination with said oncolytic agent or radiation therapy.

The present invention provides the art with a method for increasingapoptotic effects in cells and is thus effective in enhancing theanti-neoplasm effects of chemotherapies and radiation therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the dosage effect of bryostatin 1 on PKC isoforms.

FIG. 2 demonstrates the incubation time effect of bryostatin 1 on PKCisoforms.

FIG. 3 demonstrates that down regulation of PKC-β enhances the efficacyof γ-irradiation.

FIG. 4 shows that increased expression of PKC-β demonstrates resistanceto radiation stimulated cell death.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that use of PKC inhibitors,especially a particular class of protein kinase C inhibitors, reduces orinhibits anti-apoptotic effects in a cell. Consequently, such compoundscan be used to enhance the anti-neoplasm effects of chemotherapies andradiation therapies.

The method of this invention may employ any PKC inhibitor known in theart including non-specific PKC inhibitors and specific PKC inhibitors ofdifferent isoforms. Informations about PKC inhibitors, and methods fortheir preparation are readily available in the art. For example,different kinds of PKC inhibitors and their preparation are described inU.S. Pat. Nos. 5,621,101, 5,621,098, 5,616,577, 5,578,590, 5,545,636,5,491,242, 5,488,167, 5,481,003, 5,461,146, 5,270,310, 5,216,014,5,204,370, 5,141,957, 4,990,519, and 4,937,232, all of which areincoporated herein by reference. Preferably the present inventionutilizes those protein kinase C inhibitors that effectively inhibit theβ isozyme. One suitable group of compounds are generally described inthe prior art as bis-indolylmaleimides or macrocyclicbis-indolylmaleimides. Bis-indolymaleimides well recognized in the priorart include those compounds described in U.S. Pat. Nos. 5,621,098,5,552,396, 5,545,636, 5,481,003, 5,491,242, and 5,057,614, allincorporated by reference herein. Macrocyclic bis-indolylmaleimides areparticularly represented by the compounds of formula I. These compounds,and methods for their preparation, have been disclosed in U.S. Pat. No.5,552,396, which is incorporated herein by reference. In accordance withthe present invention, these compounds are administered in combinationwith other anti-neoplasm therapies to a mammal in need of suchtreatment. In particular, these compounds can be used to enhance theanti-neoplasm effects of chemotherapies and radiation therapies.

U.S. Pat. No. 5,545.636, describes PKC inhibitors of the formula:

wherein:

R¹ is

R^(1′) is hydrogen, C₁-C₄ alkyl, aminoalkyl, monoalkylaminoalkyl, ordialkylaminoalkyl;

R² and R^(2′) are independently hydrogen, alkyl, alkoxyalkyl,hydroxyalkyl, C₁-C₃ alkylthio, S(O)C₁-C₃ alkyl, CF_(3;)

R³ is hydrogen or CH₃CO-;

R^(4,) R^(4′), R⁵, R^(5′), R⁶, R^(6′), R⁷ and R^(7′) are independentlyhydrogen, halogen, alkyl, hydroxy, alkoxy, —COO(C₁-C₃ alkyl), CF₃,nitro, amino, acetylamino, monoalkylamino, dialkylamino, alkylthio,C₁-C₃ alkylthio, or S(O)C₁-C₃ alkyl;

R¹² is hydrogen, alkyl, haloalkyl, cycloalkyl, acetyl, aryl, —CH(aryl)₂,amino, monoalkylamino, dialkylamino, guanidino,—C(=N(alkoxycarbonyl))NH(alkyoxycarbonyl), amidino, hydroxy, carboxy,alkoxycarbonyl or heterocyclyl;

p and q are independently 1, 2, 3, or 4;

s is 0, 1, 2 or 3;

t is 1 or 2;

u is 0 or 1; or

pharmaceutically acceptable salts or solvates thereof.

One preferred class of compounds for use in the method of the inventionhas the formula:

wherein:

W is —O-, —S-, —SO-, —SO₂-, —CO-, C₂-C₆ alkylene, substituted alkylene,C₂-C₆ alkenylene, -aryl-, -aryl(CH₂)_(m)O-, -heterocycle-,-heterocycle-(CH₂)_(m)O-, -fused bicyclic-, -fused bicyclic—(CH₂)_(m)O-,—NR³-, —NOR³-, —CONH-, or —NHCO-;

X and Y are independently C₁-C₄ alkylene, substituted alkylene, ortogether X, Y, and W combine to form —(CH₂)_(n)—AA-;

R¹s are hydrogen or up to four optional substituents independentlyselected from halo, C₁-C₄ alkyl, hydroxy, C₁-C₄ alkoxy, haloalkyl,nitro, NR⁴R⁵, or —NHCO(C₁-C₄ alkyl);

R² is hydrogen, CH₃CO-, NH₂, or hydroxy;

R³ is hydrogen, (CH₂)_(m)aryl, C₁-C₄ alkyl, —COO(C₁-C₄ alkyl), —CONR⁴R⁵,—(C=NH)NH₂, —SO(C₁-C₄ alkyl), —SO₂ (NR⁴R⁵), or —SO₂ (C₁-C₄ alkyl);

R⁴ and R⁵ are independently hydrogen, C₁-C₄ alkyl, phenyl, benzyl, orcombine to the nitrogen to which they are bonded to form a saturated orunsaturated 5 or 6 member ring;

AA is an amino acid residue;

m is independently 0, 1, 2, or 3; and

n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt,prodrug or ester thereof.

A more preferred class of compounds for use in this invention isrepresented by formula I wherein the moieties -X-W-Y- contain 4 to 8atoms, which may be substituted or unsubstituted. Most preferably, themoieties -X-W-Y- contain 6 atoms.

Other preferred compounds for use in the method of this invention arethose compounds of formula I wherein R¹ and R² are hydrogen; and W is asubstituted alkylene, —O-, S-, —CONH-, —NHCO- or —NR³—. Particularlypreferred compounds are compounds of the formula Ia:

wherein Z is —(CH₂)_(p)- or —(CH₂)_(p)—O—(CH₂)_(p)-; R⁴ is hydroxy, —SH,C₁-C₄ alkyl, (CH₂)_(m)aryl, —NH(aryl), —N(CH₃) (CF₃), —NH(CF₃), or—NR⁵R⁶; R⁵ is hydrogen or C₁-C₄ alky; R⁶ is hydrogen, C₁-C₄ alkyl orbenzyl; p is 0, 1, or 2; and m is independently 2 or 3, or apharmaceutically acceptable salt, prodrug or ester thereof. Mostpreferred compounds of the formula Ia are those wherein Z is CH₂; and R⁴is —NH₂, —NH(CF₃), or —N(CH₃)₂.

Other preferred compounds for use in the method of the present inventionare compounds wherein W in formula I is —O-, Y is a substitutedalkylene, and X is an alkylene. These preferred compounds arerepresented by formula Ib:

wherein Z is —(CH₂)_(p)-; R⁴ is —NR⁵R⁶, —NH(CF₃), or —N(CH₃) (CF₃); R⁵and R⁶ are independently H or C₁-C₄ alkyl; p is 0, 1, or 2; and m isindependently 2 or 3, or a pharmaceutically acceptable salt, prodrug orester thereof. Most preferred compounds of formula Ib are those whereinp is 1; and R⁵ and R⁶ are methyl.

Because they contain a basic moiety, the compounds of formulae I, Ia,and Ib can also exist as pharmaceutically acceptable acid additionsalts. Acids commonly employed to form such salts include inorganicacids such as hydrochloric, hydrobromic, hydroiodic, sulfuric andphosphoric acid, as well as organic acids such as para-toluenesulfonic,methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic, succinic,citric, benzoic, acetic acid, and related inorganic and organic acids.Such pharmaceutically acceptable salts thus include sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,mono-hydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleate, 2-butyne-1,4-dioate, 3-hexyne-2, 5-dioate, benzoate,chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, hippurate, β-hydroxybutyrate, glycolate, maleate,tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate and the like. Particularly thehydrochloric and mesylate salts are used.

In addition to pharmaceutically-acceptable salts, other salts also canexist. They may serve as intermediates in the purification of thecompounds, in the preparation of other salts, or in the identificationand characterization of the compounds or intermediates.

The pharmaceutically acceptable salts of compounds of formulae I, Ia,and Ib can also exist as various solvates, such as with water, methanol,ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of suchsolvates can also be prepared. The source of such solvate can be fromthe solvent of crystallization, inherent in the solvent of preparationor crystallization, or adventitious to such solvent.

It is recognized that various stereoisomeric forms of the compounds offormulae I, Ia, and Ib may exist; for example, W may contain a chiralcarbon atom in the substituted alkylene moiety. The compounds arenormally prepared as racemates and can conveniently be used as such.Alternatively, both individual enantiomers can be isolated orsynthesized by conventional techniques if so desired. Such racemates andindividual enantiomers and mixtures thereof form part of the compoundsused in the methods of the present invention.

The compounds utilized in this invention also encompass thepharmaceutically acceptable prodrugs of the compounds of formulae I, Ia,and Ib. A prodrug is a drug which has been chemically modified and maybe biologically inactive at its site of action, but which may bedegraded or modified by one or more enzymatic or other in vivo processesto the parent bioactive form. This prodrug likely may have a differentpharmacokinetic profile than the parent, enabling easier absorptionacross the mucosal epithelium, better salt formation or solubility,and/or improved systemic stability (an increase in plasma half-life, forexample). Typically, such chemical modifications include the following:

1) ester or amide derivatives which may be cleaved by esterases orlipases;

2) peptides which may be recognized by specific or nonspecificproteases; or

3) derivatives that accumulate at a site of action through membraneselection of a prodrug form or a modified prodrug form; or anycombination of 1 to 3, supra. Conventional procedures for the selectionand preparation of suitable prodrug derivatives are described, forexample, in H. Bundgaard, Design of Prodrugs, (1985).

The synthesis of various bis-indole-N-maleimide derivatives is describedin Davis et al. U.S. Pat. No. 5,057,614 and the synthesis of thepreferred compounds suitable for use in this invention are described inthe previously identified U.S. Pat. No. 5,552,396 and in Faul et al. EPpublication 0 657 411 A1, all of which are incorporated herein byreference.

One particularly preferred protein kinase C inhibitor for use in themethod of this invention is the compound described in Example 5g((S)-3,4-[N,N′-1,1′-((2″-ethoxy)-3′″(O)-4′″-(N,N-dimethylamino)-butane)-bis-(3,3′-indolyl)]-1(H)-pyrrole-2,5-dioneHydrochloride Salt) of the aforementioned U.S. Pat. No. 5,552,396. Thiscompound is a potent protein kinase C inhibitor. It is selective toprotein kinase C over other kinases and is highly isozyme-selective,i.e., it is selective for the beta-1 and beta-2 isozymes. Other salts ofthis compound also would be favored, especially the mesylate salts.

A preferred mesylate salt can be prepared by reacting a compound of theformula II

with methanesulfonic acid in a non-reactive organic solvent, preferablyan organic/water mixture, and most preferably water-acetone. Othersolvents such as methanol, acetone, ethylacetate and mixtures thereofare operable. The ratio of solvent to water is not critical andgenerally determined by the solubility of the reagents. Preferredsolvent to water ratios are generally from 0.1:1 to 100:1 solvent towater by volume. Preferably, the ratio is 1:1 to 20:1 and mostpreferably 5:1 to 10:1. The optimal ratio is dependent on the solventselected and is preferably acetone at a 9:1 solvent to water ratio.

The reaction usually involves approximately equimolar amounts of the tworeagents, although other ratios, especially those wherein themethanesulfonic acid is in excess, are operative. The rate of additionof methanesulfonic acid is not critical to the reaction and may be addedrapidly (<5 minutes) or slowly over 6 or more hours. The reaction iscarried out at temperatures ranging from 0° C. to reflux. The reactionmixture is stirred until formation of the salt is complete, asdetermined by x-ray powder diffraction and can take from 5 minutes to 12hours.

The salts of the present invention are preferably and readily preparedas a crystalline form. The trihydrate form of the salt may be readilyconverted to the monohydrate upon drying or exposure to 20-60% relativehumidity. The salt is substantially crystalline demonstrating a definedmelting point, birefringence, and an x-ray diffraction pattern.Generally, the crystals have less than 10% amorphous solid andpreferably less than 5% and most preferably less than 1% amorphoussolid.

The mesylate salt is isolated by filtration or other separationtechniques appreciated in the art directly from the reaction mixture inyields ranging from 50% to 100%. Recrystallization and otherpurification techniques known in the art may be used to further purifythe salt if desired.

The PKC inhibitors, including the compounds described above, are used incombination with conventional anti-neoplasm therapies to treat mammals,especially humans with neoplasia. The procedures for conventionalanti-neoplasm therapies, including chemotherapies, e.g. using oncolyticagents and radiation therapies e.g., γ-irradiation are known, readilyavailable, and routinely practiced in the art, e.g., see Harrison'sPRINCIPLES OF INTERNAL MEDICINE 11th edition, McGraw-Hill Book Company.

Neoplasia is characterized by abnormal growth of cells which oftenresults in the invasion of normal tissues, e.g., primary tumors or thespread to distant organs, e.g., metastasis. The treatment of anyneoplasia by conventional anti-neoplasm therapies can be enhanced by thepresent invention. Such neoplastic growth includes but not limited toprimary tumors, primary tumors that are incompletely removed by surgicaltechniques, primary tumors which have been adequately treated but whichare at high risk to develop a metastatic disease subsequently, and anestablished metastatic disease.

Specifically, the PKC inhibitors described above can enhance theanti-neoplasm effects of an oncolytic agent. The wide variety ofavailable oncolytic agents are contemplated for combination therapy inaccordance with present invention. In a preferred embodiment, oncolyticagents that assert their cytotoxic effects by activating programmed celldeath or apoptosis are used in combination with the described PKCinhibitors. These include but not limited to1-β-D-arabinofuranosylcytosine or Ara-c, etoposide or VP-16,cis-diamminedichloroplatinum (II) or cis-platinum, doxorubicin oradriamycin, 2-chloro-2-deoxyadenosine, 9-β-D-arabinosyl-2-fluoroadenine,and glucocorticoids.

All the neoplastic conditions treatable with such oncolytic agents canbe treated in accordance with the present invention by using acombination of a PKC inhibitor with one or more oncolytic agents. Theoncolytic agents assert the cytotoxicity or anti-neoplasm effects in avariety of specific neoplastic conditions. For example, Ara-c isnormally used for treatment of childhood-null acute lymphoid leukemia(ALL), thymic ALL, B-cell ALL, acute myeloid leukemia, acutegranulocytic leukemia and its variants, non-Hodgkins lymphoma,myelomonocytoid leukemia, acute megakaryocytoid leukemia and Burkitt'slymphoma, Adult-B-ALL, acute myeloid leukemia, chronic lymphoidleukemia, chronic myeloid leukemia, and T cell leukemia. VP-16 isnormally used for treatment of testicular carcinoma, small and largenon-small cell lung carcinoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma, choriocarcinoma, Ewing's sarcoma, and acute granulocyticleukemia. Cis-platinum can be employed for treatment of testicularcarcinoma, germ cell tumors, ovarian carcinomas, prostate cancer, lungcancer, sarcomas, cervical cancer, endomermetrial cancer, gastriccancer, breast cancer, and cancer of the head and neck.2-Chloro-2-deoxyadenosine and 9-β-D-arabinosyl-2-fluoroadenine can beused to treat chronic lymphoid leukemia, lymphomas and hairy cellleukemia. Doxorubicin can be used to treat acute granulocytic leukemiaand its variants, ALL, breast cancer, bladder cancer, ovarian cancer,thyroid cancer, lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma,sarcomas, gastric carcinoma, prostate cancer, endometrial cancer, Wilm'stumor and neuroblastoma. Clinical effects of oncolytic agents in allneoplastic conditions treatable with oncolytic agents including the onesdiscussed above can be potentiated by use of a combination therapy withthe identified PKC inhibitors in accordance with the present invention.

The PKC inhibitors identified in the present invention can also enhancethe anti-neoplasm effects of a radiation therapy. Usually γ-irradiationis used to treat the site of a solid tumor directly.

Experimental results provided in the present invention demonstrate thatthe complete down regulation or loss of protein kinase C-β is associatedwith the synergistical enhancement of the oncolytic induced apoptosis inhuman leukemic cells (FIG. 1). Similarly, significant down regulation ofprotein kinase C-β in U937 human leukemic cells enhances radiationstimulated cell death (FIG. 2). U937 human leukemic cells thatoverexpress protein kinase C-β demonstrate resistance to radiationstimulated cell death (FIG. 3). These data provide a strong indicationthat the PKC inhibitors, especially β isozyme selective inhibitors,preferably used in accordance with the present invention can enhancetumor killing or the anti-neoplasm effects of chemotherapies andradiation therapies and improve clinical responses to these currentlyused therapeutic modalities.

The PKC inhibitors of the present invention are administered incombination with other anti-neoplasm therapies including oncolyticagents and radiation therapy. The phrase “in combination with othertherapies” means that the compounds can be administered shortly before,shortly after, or concurrent with such other anti-neoplasm therapies.The compounds can be administered in combination with more than oneanti-neoplasm therapy. In a preferred embodiment, the compounds areadministered from 2 weeks to 1 day before any chemotherapy, or 2 weeksto 1 day before any radiation therapy. Alternatively, the PKC inhibitorscan be administered during chemotherapies and radiation therapies. Ifadministered following chemotherapy or radiation therapy, the PKCinhibitors should be given within 1 to 14 days following the primarytreatments.

One skilled in the art will recognize that the amount of PKC inhibitorto be administered in accordance with the present invention incombination with other anti-neoplasm agents or therapies is that amountsufficient to enhance the anti-neoplasm effects of oncolytic agents orradiation therapies or that amount sufficient to induce apoptosis orcell death. Such amount may vary inter alia, depending upon the size andthe type of neoplasia, the concentration of the compound in thetherapeutic formulation, the specific anti-neoplasm agents used, thetiming of the administration of the PKC inhibitors relative to the othertherapies, and the age, size and condition of the patient.

Both in vivo and in vitro tests can be used to assess the amount of thecompounds needed for inducing apoptosis. For example, human leukemiccells could be exposed in vitro to various concentrations of oncolyticagents, e.g., Ara-c, or to radiation in the presence or absence of thePKC inhibitor compounds used in the present invention. Appropriateneoplastic cell types can be chosen for different oncolytic agents.Other protein kinase C selective inhibitors can also be used forcomparison. At various time points, cells would be examined forviability by conventional methods or by any means available in the art.Apoptosis or cell death can be measured by any means known in the art.Cell death can be determined and quantified via trypan blue exclusion,and reduced clonogenecity in soft agar. As well understood by thoseskilled in the technology, apoptosis is a specific mode of cell deathrecognized by a characteristic pattern of morphological, biochemical,and molecular changes including but not limited to, endonucleolysis (DNAladder), abnormal DNA breaks, and condensation of chromatin andcytoplasm (condensed and punctate nuclei). These changes can be readilydetected by any means known in the art, e.g., microscopy; flowcytometric methods based on increased sensitivity of DNA to denaturationand altered light scattering properties; DNA fragmentation as assessedby agarose gel electrophoresis; terminal DNA transferase assay, (TdTassay), and nick translation assay (NT assay).

In vivo studies can be done using tumor xenografts inoculated intoimmunocompromised or sygenic animals. After inoculation and growth ofthe primary implant, the animals would be treated with the compounds inthe present invention prior to exposure to the desired oncolytic orradiation treatment. The size of the tumor implant before and after eachtreatment in the presence and absence of the compounds in the presentinvention can be used as an indication of the therapeutic efficacy ofthe treatment.

Generally, an amount of protein kinase C inhibitor to be administered incombination with other anti-neoplasm therapies is decided on a case bycase basis by the attending physician. As a guideline, the extent of theneoplasia, the body weight, and the age of the patient will beconsidered, among other factors, when setting an appropriate dose.Normally, the PKC inhibitors of the present invention are expected topotentiate the anti-neoplasm effects of oncolytic agents and radiationtherapy from about 2 fold to about 10 fold.

Generally, a suitable dose is one that results in a concentration of theprotein kinase C inhibitor at the site of tumor cells in the range of0.5 nM to 200 μM, and more usually from 20 nM to 80 nM. It is expectedthat serum concentrations of 40 nM to 150 nM should be sufficient inmost circumstances.

To obtain these treatment concentrations, a patient in need of treatmentlikely will be administered between about 0.1 mg per day per kg of bodyweight and 1.5 mg per day per kg. Usually, not more than about 1.0 mgper day per kg of body weight of protein kinase C inhibitor should beneeded. As noted above, the above amounts may vary on a case-by-casebasis.

The compounds of formula I and the preferred compounds of formula Ia andIb are preferably formulated prior to administration. Suitablepharmaceutical formulations are prepared by known procedures using wellknown and readily available ingredients. In making the compositionssuitable for use in the method of the present invention, the activeingredient will usually be mixed with a carrier, or diluted by acarrier, or enclosed within a carrier which may be in the form of acapsule, sachet, paper or other container. When the carrier serves as adiluent, it may be a solid, semisolid or liquid material which acts as avehicle, excipient or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosol (as a solid or in a liquid medium), soft and hard gelatincapsules, suppositories, sterile injectable solutions and sterilepackaged powders for either oral or topical application.

Some examples of suitable carriers, excipient, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphates, alginate, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propylhydroxybenzoates, talc,magnesium stearate and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents or flavoring agents. Thecompositions of the invention may be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient. The compositions are preferablyformulated in a unit dosage form, each dosage containing from about 0.05mg to about 3 g, more usually about 64 mg of the active ingredient.However, it will be understood that the therapeutic dosage administeredwill be determined by the physician in the light of the relevantcircumstances including the severity of the condition to be treated, thechoice of compound to be administered and the chosen route ofadministration. Therefore, the above dosage ranges are not intended tolimit the scope of the invention in any way. The term “unit dosage form”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalcarrier.

In addition to the above formulations, most of which may be administeredorally, the compounds used in the method of the present invention alsomay be administered topically. Topical formulations include ointments,creams and gels.

Ointments generally are prepared using either (1) an oleaginous base,i.e., one consisting of fixed oils or hydrocarbons, such as whitepetrolatum or mineral oil, or (2) an absorbent base, i.e., oneconsisting of an anhydrous substance or substances which can absorbwater, for example anhydrous lanolin. Customarily, following formationof the base, whether oleaginous or absorbent, the active ingredient(compound) is added to an amount affording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internalphase), comprising typically fixed oils, hydrocarbons, and the like,such as waxes, petrolatum, mineral oil, and the like, and an aqueousphase (continuous phase), comprising water and any water-solublesubstances, such as added salts. The two phases are stabilized by use ofan emulsifying agent, for example, a surface active agent, such assodium lauryl sulfate; hydrophilic colloids, such as acacia colloidalclays, veegum, and the like. Upon formation of the emulsion, the activeingredient (compound) customarily is added in an amount to achieve thedesired concentration.

Gels comprise a base selected from an oleaginous base, water, or anemulsion-suspension base. To the base is added a gelling agent whichforms a matrix in the base, increasing its viscosity. Examples ofgelling agents are hydroxypropyl cellulose, acrylic acid polymers, andthe like. Customarily, the active ingredient (compounds) is added to theformulation at the desired concentration at a point preceding additionof the gelling agent.

The amount of compound incorporated into a topical formulation is notcritical; the concentration should be within a range sufficient topermit ready application of the formulation to the affected tissue areain an amount which will deliver the desired amount of compound to thedesired treatment site.

The customary amount of a topical formulation to be applied to anaffected tissue will depend upon an affected tissue size andconcentration of compound in the formulation. Generally, the formulationwill be applied to the effected tissue in an amount affording from about1 to about 500 μg compound per cm² of an affected tissue. Preferably,the applied amount of compound will range from about 30 to about 300μg/cm², more preferably, from about 50 to about 200 μg/cm², and, mostpreferably, from about 60 to about 100 μg/cm².

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way.

Formulation I Hard gelatin capsules are prepared using the followingingredients: Quantity (mg/capsule) Active agent 250 starch, dried 200magnesium stearate 10 Total 460 mg

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

Formulation 2 A tablet is prepared using the ingredients below: Quantity(mg/capsule) Active agent 250 cellulose, microcrystalline 400 silicondioxide, fumed 10 stearic acid 5 Total 665 mg

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3 Tablets each containing 60 mg of active ingredient aremade as follows: Quantity (mg/tablet) Active agent 60 mg starch 45 mgmicrocrystalline cellulose 35 mg polyvinylpyrrolidone (as 10% solutionin water) 4 mg sodium carboxymethyl starch 4.5 mg magnesium stearate 0.5mg talc 1 mg Total 150 mg

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

EXAMPLES Example 1

Effects of Bryostatin to PKC isoforms

This experiment demonstrates the dosage and time effects of bryostatinto PKC isoforms.

Human leukemia cells U937 in the amount of 0.5×10⁶ were treated withvarious amount of bryostatin 1 for 24 hours. Subsequently, the cellswere solubilized for preparation of protein samples according to aroutine procedure. The protein samples from bryostatin treated cellswere then used in Western blot analysis with a protein kinase C-βspecific antiserum previously described in Ways et al., Cell Growth &Differentiation 1994, 5: 1195-1203. As shown in FIGS. 1 and 2,bryostatin treatment caused PKC-β activity to decrease within certainamount of time, i.e., 10 nM bryostatin affects PKC-β within 2 hours, or1 nM bryostatin affects PKC-β within 24 hours. In a repeated experiment,similar results were obtained.

Example 2

The enhanced efficacy of γ-irradiation caused by PKC-β down regulation

This experiment demonstrates that PKC-β down regulation enhances theefficacy of γ-irradiation.

Human leukemia cells U937 were treated for 24 hours with either 3 nMbryostatin 1 or the control solution, i.e., the vehicle forbryostatin 1. The cells were then irradiated with either 500 or 1000rads of γ-irradiation. Seventy-two hours after irradiation, cellularviability was examined using propidium iodide exclusion and quantifiedby FACS analysis as previously described in Ways et al., Cell Growth &Differentiation 1994, 5: 1195-1203. Viability assays were performed intriplicate. As shown in FIG. 3, γ-irradiation-induced apoptosis wasenhanced under the condition when PKC-β was significantly down-regulatedusing bryostatin 1. Similar results were obtained in several repeatedexperiments.

Example 4

Cells Overexpressing PKC-β Demonstrate Resistance to RadiationStimulated Cell Death

Parental U937 cells and U937 PKC-ξ overexpressing cells (PKC-ξ cells)were treated with 0, 500, or 1000 rads of γ-irradiation. It is knownthat PKC-ξ cells display increased level of PKC-β (Ways et al., CellGrowth & Differentiation, 1994, 5:1195-1203). Seventy two hours afterirradiation, cellular viability was examined using propidium iodideexclusion and quantified by FACS analysis as previously described inWays et al., Cell Growth & Differentiation, 1995, 6: 371-382. Viabilityassays were performed in triplicate. As shown in FIG. 4, cells having anincreased level of PKC-β demonstrated resistance to radiation stimulatedcell death. Similar results were obtained in several repeatedexperiments.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, since theyare to be regarded as illustrative rather than restrictive. Variationsand changes may be made by those skilled in the art without departingfrom the spirit of the invention.

What is claimed is:
 1. A method for treating a neoplastic condition sensitive to the combination below which comprises administering to a mammal in need of such treatment an effective amount of an oncolytic agent having an anti-neoplastic effect in combination with a protein kinase C inhibitor selective for a beta-1- or beta-2- isozyme of protein kinase C, wherein the protein kinase C inhibitor is administered in an amount sufficient to enhance the anti-neoplastic effect of the oncolytic agent, and wherein the protein kinase C inhibitor is a bis-indolylmalemide and has the following formula:

wherein:

R¹ is R^(1′) is hydrogen, C₁-C₄ alkyl, aminoalkyl, monoalkylaminoalkyl, or dialkylaminoalkyl; R² and R^(2′) are independently hydrogen, alkyl, alkoxyalkyl, hydroxyalkyl, C₁-C₃ alkylthio, S(O)C₁-C₃ alkyl, or CF₃; R³ is hydrogen or CH₃CO-; R⁴, R^(4′), R⁵, R^(5′), R⁶, R^(6′), R⁷ and R^(7′)are independently hydrogen, halogen, alkyl, hydroxy, alkoxy, —COO(C₁-C₃ alkyl), CF₃, nitro, amino, acetylamino, monoalkylamino, dialkylamino, alkylthio, C₁-C₃ alkylthio, or S(O)C₁-C₃ alkyl; R¹² is hydrogen, alkyl, haloalkyl, cycloalkyl,acetyl, aryl, —CH(aryl)₂, amino, monoalkylamino, dialkylamino, guaidino, —C(=N(alkoxycarbonyl))NH(alkyoxycarbonyl), amidino, hydroxy, carboxy, alkoxycarbonyl or heterocyclyl; p and q are independently 1, 2, 3, or 4; s is 0, 1, 2 or 3; t is 1 or 2; u is 0 or 1; or a pharmaceutically acceptable salt or solvate thereof. 